I am in need of suggestions for overcoming a major obstacle
in Arabidopsis Agrobacterium-mediated transformation. I am
a novice working in a lab where there is a lot of experience
with tissue culture and transformation of tobacco, not
Arabidopsis. I have been following published protocols for
use of root explants, and have been using the same vectors
that have been used successfully in our lab to transfer
kanamycin resistance to tobacco. My control roots produce
lots of green callus and shoots, but the roots placed on
50mg/L kanamycin show only yellow callus and stop growing -
apparently they have not been transformed. I have varied
the pre-transformation time on callus inducing medium from
3 days to 7 days.
I would appreciate any ideas someone would be willing
to provide. I will provide more details as needed. Thanks
in advance for any help!!!
Sue Bard - University of Maryland, College Park and
Howard Community College, Columbia, Md.